Systems and methods for providing microfluidic devices

ABSTRACT

A microfluidic valve system is disclosed that includes a matrix, a hydrophilic acceptor region a hydrophilic transfer region, and a hydrophobic gap between the acceptor region and the transfer region.

PRIORITY

This application is a divisional application of U.S. application Ser.No. 13/625,510 filed on Sep. 24, 2012, which claims priority to U.S.Provisional Patent Application Ser. No. 61/538,255 filed Sep. 23, 2011,the entire disclosures of which are hereby incorporated by reference intheir entireties.

GOVERNMENT SUPPORT

This work is supported by the National Science Foundation under GrantNo. NSF-OISE-0530203, the U.S. government has certain rights to thisinvention.

BACKGROUND

Self-contained paper microfluidic devices can provide inexpensive newtools for rapid diagnostic information in diverse applications such asthe healthcare of an individual from a biological fluid or a hazardouschemical in an environment liquid sample. Advantageous elements of paperbased microfluidic devices relative to traditional laboratory baseddiagnostics include the ease of use for individual, rapid diagnostics,lack of sophisticated support equipment, simplified assessment ofdiagnostic result, low cost, and disposability to prevent contamination.

Since the 1990s, many advances have been made regarding the developmentof two-dimensional media based microfluidics for the detection ofanalytes using a variety of fluidic samples. Typically, paper basedmedia, referred to as microfluidic paper analytical devices (MPAD), havebeen patterned by layering hydrophobic chemistries on hydrophilic mediacreating physical barriers to contain wicking or capillary fluidicmotion. To create microchannel barriers, a variety of technologies andchemistries have been employed.

For example, PCT Patent Application Publication No. WO 2010/022324discloses methods of patterning hydrophobic materials onto hydrophilicsubstrates as well as methods of impregnating hydrophilic substrateswith a hydrophobic material. U.S. Patent Application Publication No.2009/0298191 discloses methods of patterning porous media to providelateral flow and flow-through bioassay devices wherein the devicesinclude a porous, hydrophilic medium and a fluid impervious barriercomprising a polymerizable photoresist, with the barrier substantiallypermeating the thickness of the porous, hydrophilic medium and defininga boundary of an assay region (containing an assay reagent) within theporous, hydrophilic medium. Other developments have used polystyrene,wax-based and superhydrophobic patterning processes to form physicalmicrochannel barriers defining hydrophilic channels or regions.

U.S. Patent Application Publication No. 2011/0123398 disclosesthree-dimensional microfluidic devices that include a plurality ofpatterned porous, hydrophilic layers and a fluid-impermeable layerdisposed between adjacent patterned porous, hydrophilic layers. Eachpatterned porous, hydrophilic layer is disclosed to include afluid-impermeable barrier that substantially permeates the thickness ofthe porous, hydrophilic layer and defines boundaries of one or morehydrophilic regions within the patterned porous, hydrophilic layer. Thefluid-impermeable layer has openings that are aligned with at least partof the hydrophilic region within at least one adjacent patterned porous,hydrophilic layer.

U.S. Patent Application Publication No. 2008/0025873 disclosesmicrofluidic devices that include a substrate and a non-valve capillarymechanism, as well as a reservoir and one or more channels leading tothe reservoir, wherein the non-valve capillary mechanism is within thereservoir, and prevents fluid delivered to the reservoir from wickingfrom the reservoir into the channels. A delivered fluid ishydrophilically attracted to and retained within the reservoir.

In other devices, processes employed to delay fluidic motion have beenbased on abruptly changing the physical geometry of the microchannelsthrough enlargement of the microchannel. Assembling two or more multipledelay valves to form a joined region where at least two fluids wererequired to advance the fluid created a temporary trigger valve having alonger delay time. In still other devices, paraffin wax has been used torestrict wicking through a control point between layers.

Although these devices may prevent undesired mixing of fluids betweenreservoirs and adjacent channels, the need remains for the ability tocontrol mixing of fluids with a microfluidic valve that does not employmechanical or electrical mechanisms to control the valve therebyrestricting the utility of the device and its stand alone use.

SUMMARY

In accordance with an embodiment, the invention provides a microfluidicvalve system that includes a matrix, a hydrophilic acceptor region ahydrophilic transfer region, and a hydrophobic gap between the acceptorregion and the transfer region.

In accordance with an embodiment, the invention provides a microfluidicnon-mechanical valve that includes a hydrophobic material permeating thethickness of hydrophilic media defining a hydrophobic channel separatinga hydrophilic transfer region containing a transfer agent and ahydrophilic acceptor region, wherein the microfluidic non-mechanicalvalve is opened by wetting the transport agent hydrophilic stagingregion allowing fluid movement across the hydrophobic gap between thehydrophilic transfer region and the hydrophilic acceptor region.

In accordance with an embodiment, the invention provides a method ofmaking a microfluidic valve on a matrix, comprising the hydrophilicacceptor region and a hydrophilic transfer region. The method includesthe step of containing a transfer agent separated by a hydrophobic gap,wherein a transfer agent is deposited on hydrophilic transfer region.

In accordance with various further embodiments of the present invention,a non-mechanical valve is provided that may be opened solely by usingmicrofluidic properties contained within the device upon application ofa liquid sample to be tested.

In accordance with certain embodiments, a physical hydrophobic barriermay be created by applying hydrophobic materials, including, but notlimited to, photoresist, polystrene, PDMS and waxes on a hydrophilicmatrix that define hydrophilic regions including, but not limited to,microchannels and reservoirs.

The term valve or microfluidic valve or diode refers herein to anon-mechanical device to control the flow of a fluid created bypositioning a hydrophobic region between two hydrophilic regions. Avalve is constructed by placing a transfer agent, such as a surfactant,on the hydrophilic region that controls opening the valve. The valve isopened when a fluid solubilizes the surfactant allowing fluid to passthrough the hydrophobic region to the acceptor hydrophilic region. Inthis manner, the value operates in only one direction. Once opened,fluid flow is able to go in both directions. The arrows in the figuresindicate the fluid flow of the valve.

In accordance with further embodiments, virtual hydrophobic barrier iscreated by altering the surface wettability properties of the matrixthat define hydrophobic regions and hydrophilic regions including, butnot limited to, microchannels and reservoirs. The surface wettabilityproperties relate to rendering the matrix to be more conducive to fluidmovement.

BRIEF DESCRIPTION OF THE DRAWINGS

The following description may be further understood with reference tothe accompanying drawings in which:

FIG. 1 shows an illustrative diagrammatic view of a process ofphotopatterning layered paper in accordance with an embodiment of theinvention using a photo-initiator;

FIGS. 2A-2C show an illustrative microphotographic views of ahydrophilic pattern on a hydrophobic surface, the contact angle of awater droplet on the hydrophobic surface, and a cross-sectional viewalong the line 2C-2C of FIG. 2A;

FIGS. 3A-3C show illustrative diagrammatic views of photo-patternedhydrophilic channels with decreasing width, hydrophobic gaps withdecreasing widths, and a graphical representation of designed widthverses reproduced width;

FIGS. 4A-4D show illustrative diagrammatic views of an S-shaped channelproduced in accordance with an embodiment of the invention, aphotomicrographic taken along line 4B-4B of FIG. 4A, time-sequencephotomicrographs on a tri-ethylene glycol (TEG)-grafted surface, andtime-sequence photomicrographs on a MU tri-ethylene glycol(MUTEG)-grafted surface;

FIGS. 5A-5D show illustrative diagrammatic views of a non-mechanicalmicrofluidic valve in accordance with an embodiment of the invention, amicroscopic schematic of the dashed area shown in FIG. 5A,time-sequential photographs showing the fluid in two reverselyconfigured valves, and photograph of a non-mechanical microfluidic valvewith human blood serum;

FIGS. 6A-6B show illustrative diagrammatic views of a trigger valve inaccordance with an embodiment of the invention, and time-sequentialphotographs showing gated fluid released by a triggering of fluid;

FIGS. 7A-7B show illustrative diagrammatic views of a delay valve inaccordance with an embodiment of the invention, and time-sequentialphotographs of the synchronized wicking of fluid in two delay valves;

FIGS. 8A-8B show illustrative diagrammatic views of a sequential loadingsystem in accordance with an embodiment of the invention, andtime-sequential photographs of fluid A and fluid B moving in such asystem;

FIGS. 9A-9G show illustrative diagrammatic views of three-dimensionalpaper-based microfluidic devices in accordance with embodiments of theinvention, as well as a scanned cross-sectional view taken along line9G-9G of FIG. 9F;

FIGS. 10A-10C show illustrative diagrammatic views of a representativeone way valve, layers of a device in accordance with an embodiment ofthe invention, and top and bottom layers thereof;

FIGS. 11A-11D show illustrative diagrammatic views of a fluidic triggervalve in accordance with an embodiment of the invention, layers of sucha device, and top and bottom layers thereof;

FIGS. 12A-12C show illustrative diagrammatic views of a fluidic delayvalve in accordance with an embodiment of the invention, layer of such adevice, and top and bottom layers thereof;

FIGS. 13A-13D show illustrative diagrammatic views of a sequentialloading valve in accordance with an embodiment of the invention, layersof such a device, top layers thereof at three consecutive times (as wellas intensity graphs thereof), and a size comparison view;

FIG. 14 shows an illustrative diagrammatic view of a three-dimensionaldevice in accordance with a further embodiment of the present invention;

FIGS. 15A-15B show an illustrative diagrammatic views of electricalcomparison schematic diagrams of device circuits in accordance withfurther embodiment of the present invention;

FIGS. 16A-16B show illustrative diagrammatic views of a fluidic valve inaccordance with an embodiment of the invention, and a device including areservoir;

FIGS. 17A-17B show illustrative diagrammatic views of a further fluidicvalve coupled to a reservoir in accordance with a further embodiment ofthe invention, as well as a schematic illustration of a manufacturingprocessing step in forming the fluidic valve;

FIGS. 18A-18B show illustrative diagrammatic views of further fluidicvalves coupled to a reservoir showing loading and directional flow, aswell we a schematic illustration of a manufacturing processing step informing the fluidic valve; and

FIGS. 19A-19B show illustrative diagrammatic views of a fluidic valveusing enzymatic detection showing the enzyme, the substrate and thedetection spot, and five such detection assays.

DETAILED DESCRIPTION

In accordance with an embodiment, the invention provides a microfluidicvalve that is opened without any use of mechanical or physicalmechanisms. The microfluidic valve contains a transfer agent, such as asurfactant, that is deposited in a selected hydrophilic region and thatwill serve as a mechanism to open a valve allowing fluidic transfer froma hydrophilic region across a hydrophobic gap or channel to anotherhydrophilic region. The valve can be patterned using different methodson mediums together with channels and input or output terminals. Inaccordance with certain embodiments, the present invention teaches avariety of more complex valves such as delay valves and trigger valvesto provide a versatility of the desired time for fluids to be releasedor mixed. Combinations of valves were configured into two-dimensional(2D) sequential devices that were capable of exchanging two or morefluids. A further improvement of sequential devices, valves wereconfigured into three-dimensional (3D) sequential valves to transferfluids in three dimensions between 2 or more layers using multiplefluids as required for more complex diagnostic capabilities and reducingthe size of the device.

Prior micro-fluidic devices have employed various hydrophobic materials,including photoresist, polystrene, PDMS and waxes to pattern the surfaceof a paper matrix to form physical solid microchannels for paper basedmicrofluidics. In accordance with certain embodiments of the presentinvention, the applicants departed from applying hydrophobic materialsto form physical hydrophobic barriers on the surface of the papermatrix, and instead provide a novel approach where the surfaceconstitution of its cellulose fibers is covalently modified intohydrophilic or hydrophobic regions thereby creating virtual walls formedby patterned wettability of paper.

In one scheme, the initial step was to alter the surface properties ofcellulose from having terminal hydroxyl groups, hydrophilic, to terminalvinyl groups, hydrophobic. Once the hydrophobic surface was created,hydrophilic areas were patterned onto the hydrophobic vinyl surfaceusing reactive thiol-ene click chemistry and were activated using UVlight. Only those areas exposed to UV light were grafted covalently,thereby changing their surface properties from hydrophobic tohydrophilic resulting in an easily patterned surface containing virtualwalls as a novel alternative to traditional physical hydrophobic walls.

For example, FIG. 1A shows diagrammatically (at 10), cellulose fiberhaving exposed oxygen hydrogen atoms (OH) that are then combined withsilicone tri-chlorine (Si—Cl₃) as shown at 12 to produce cellulose fiberhaving molecular exposures of silicone (Si) as shown, in an hydrochloricacid (HCl) as shown at 14. As shown at 16, when a photomask is appliedto a portion of the cellulose fiber (as shown at 16) and UV light isimpinged on the exposed regions, some areas remain hydrophobic (as shownat 18), while others again become hydrophilic (as shown at 19).

A surprising result of this approach is that the lack of a physicalbarrier to create hydrophilic regions provides more flexibility infabricating new processes and utilities for paper based microfluidicssuch as a microfluidic non-mechanical valve described herein. It is alsotaught that the paper based microfluidic application or device may use avirtual barrier region in conjunction with a physical barrier region toform yet more complex applications and devices.

The chemistries used to produce patterned wettability in a poroussubstrate matrix depend on initial surface properties of the matrix,coupling agents that link a hydrophilic or hydrophobic terminal to thesurface, and the patterning methods. The substrate matrix is not limitedto paper and either hydrophilic or hydrophobic porous substrates may beused. Hydrophilic porous substrates include cellulose, glassmicrofibers, cotton, wool, silk, and other hydrophilic porous materials.Hydrophobic porous substrates include polyvinylidene fluoride, nylon,nitrocellulose, polytetrafluoroethylene, mixed cellulose ester, andother hydrophobic porous materials. For the hydrophilic substrates,printing or stamping a solution of the coupling reagents containinghydrophobic terminals may be employed to form a desired hydrophobic orhydrophilic pattern.

Alternatively, the hydrophilic substrates may be first converted to beuniformly hydrophobic by the coupling reagents, and subsequently thehydrophobic terminal of the coupling reagents may be further coupled andpatterned with another molecule to introduce hydrophilic terminals. Acoupling reagent is a molecule that has at least one functional terminalthat covalently bonds to the substrate. Examples of functional terminalsinclude trichlorosilane and trimethoxysilane, which react with hydroxylgroups of the substrate. Once the coupling reagent bonds to thesubstrate, its terminal group determines the local wettability.Terminals may be either hydrophobic including alkanes and fluorocarbonsor hydrophilic including hydroxyl and polyethylene glycol (PEG).Examples of coupling chemistry include thiol-ene click chemistry andazide alkyne Huisgen cycloaddition.

FIG. 2A shows at 20 a hydrophilic channel on a hydrophobic porous paper22, and FIG. 2C shows a cross-sectional view of the paper 22 along theline 2C-2C thereof. FIG. 2B shows a water droplet 24 on a portion of thehydrophobic surface 22. FIG. 3A shows at 30 hydrophilic channels withdecreasing width to the right. FIG. 3B shows at 32 decreasing gap widthsof about 2 mm, 1.5 mm, 1.0 mm, and 0.5 mm. FIG. 3C shows at 34 acorrelation of designed width verses reproduced width in mm, as well asdistance of reproduced width shifts from the designed width of 1.5 mm ofthe functioning gap in FIG. 3B as shown at 36.

FIG. 4A shows at 40 an S-shaped channel produced in accordance with anembodiment of the invention, and FIG. 4B shows at 42 a cross-sectionalarea of FIG. 4A taken along line 4B-4B thereof. FIG. 4C shows at 44 atime sequence (t=0 s, t=102 s and t=202 s) on a tetra (ethylene glycol)grafted surface, and FIG. 4D shows at 46 a time sequence (t=0 s, t=0.6 sand t=1.2 s) on an MU tetra (ethylene glycol) grafted surface.

In a second aspect of the invention, the applicants have resolved priorconstraints in developing a self-contained microfluidic diagnosticdevice that is able to hold or prevent passive microfluidic transfer orwicking until such time the transfer or wicking is desired. In the past,microfluidic devices that are able to delay or facilitate microfluidtransfer from one region to another, typically through a microchannel,were limited by requiring external equipment such as capillary pumps,electronics or other devices or physical structures in the microchannelsas described previously herein. The present invention does not requireany of these additional equipment or physical structures to stop ordelay microfluid transfer from one region to another.

In accordance with an embodiment of the present invention, a transferagent is deposited or applied in a selected hydrophilic region that willserve as a mechanism to open a valve allowing fluidic transfer from thishydrophilic region across a hydrophobic gap or channel to anotherhydrophilic region. The area where this action occurs is referred to asa hydrophilic transfer region or microfluidic non-mechanical valve. Themicrofluidic non-mechanical valve is opened when a fluid is applied ordelivered into the hydrophilic transfer region and the transfer agent,such as a surfactant, is solubilized or dissolved in the fluid and theagent alters the wettability of adjoining hydrophobic area allowing thefluid to transfer to the other hydrophilic region.

For example, FIG. 5A shows at 50 a representative schematic of a valveof the invention, and at 52 an embodiment of such a microfluidic valve.In particular, the valve 52 includes an anode 54 and a cathode 56, witha gap 652 between a circular portion 60 of the anode 54, and a largercircular portion 58 of the cathode 56. As further shown at 64, whenwater contacts a surfactant 66, cellulose fiber permits the liquid totravel across the hydrophobic gap as shown, with the proceeding of themeniscus shown by arrows at 72. FIG. 5C shows the one way directionalityof the valve, wherein two valves 76, 78 are coupled to a receiving area80. One valve (76) is oriented to permit fluid to reach the spot 82,while the other valve (78) is positioned in a the opposite orientation,and does not permit fluid to reach the spot 84 as shown in the timesequence photographs shown in at 74 in of FIG. 5C. FIG. 5 D shows at 86another non-mechanical microfluidic device of the invention with humanblood serum.

The surprising and novel feature of this valve is that it is directionalin function. The valve is not opened when the fluid enters into thehydrophilic acceptor region that does not contain the transfer agent(e.g., valve 78 of FIG. 5C). This function allows a fluid to be held inthe reservoir until the fluid is to be released. Transfer agents includesurfactants that are either nonionic or ionic surfactants. Ionicsurfactants include anionic, cationic and Zwitterionic surfactants.Examples of nonionic surfactants include polyoxyethylene glycol alkylether, polyoxypropylene glycol, alkyl ether, polyoxyethylene glycolsorbitan alkyl ester (polysorbate), polysorbate 20 (Tween 20),polyoxyethylene glycol octylphenol ether (Triton X-100), glycerin,polyoxyethylene glycol alkylphenol ether, polyvinyl alcohol,polysorbate, glycerol alkyl ester, polyvinylpyrrolidone, polyethyleneglycol, glucoside alkyl ether and other nonionic surfactants.

In the third aspect of the present invention, the non-mechanical valvecan be used to construct complex diagnostic devices requiring the use ofmultiple diagnostic agents and steps to perform the desired assay.Assembling valves and valve variants in an array of sequential-loadingsteps is a powerful tool for performing complicated biological assays.As one example of a multiple step diagnostic assay is one that requirestwo antibodies to recognize an infectious disease where the firstantibody binds to a specific epitope on the infectious microbe, such asa pathogenic virus, bacterium or fungi, as a trap bound to paper, andthen a second antibody coupled to an indicator agent binds to theantibody trapped infectious microbe. In one set of applications thatindicator agent may be visible to the naked eye directly under normal orUV exposed light or indirectly if the indicator is only visible upon asecondary reaction. These steps may require incubation times to be fullyreactive, such as antibody binding or the development of a coloredanalyte using an enzymatic reaction. Other detection systems includeemission of fluorescence, phosphorescence or luminescence. In yet otherembodiments, systems requiring equipment for detection can use optical,magnetic, radiological, and electrical indicators. In some cases, thedetection equipment may be portable and can be linked to a diagnosticcenter via a communication link, either satellite, wireless, or directlyto the internet, that is able to perform the analysis based on thedetection of the analyte.

To construct a device having sequential-loading steps it was necessaryto design more complex valves. Two such embodiments are a trigger valveand a delay valve. In designing a trigger valve, the valve provides fora fluid to mix with a liquid sample or another fluid in a timed period.In contrast to previous devices that require support equipment toperform mixing, in the present invention the fluid to be mixed is helduntil it is released by the liquid sample or fluid to undergo mixing.The length of the channel can be adjusted to control the time forrelease of the liquid sample.

FIG. 6A shows at 90 a schematic view of such a device, and FIG. 6B showstwo valves 92, 94, one of which is coupled to a path that includes adelay element 100. As shown in the time sequence photographs (t=15 s,t=152 s, t=183 s and t=553 s), the delay unit 100 causes fluid to reachthe spot 102 well prior to the time when fluid will reach the spot 104.The trigger valves can be used in a parallel or a series array dependingupon the desired mixing reactions.

In another configuration, a delay valve is provided, which may be usedto delay the release of a fluid by the length of the channel, denotedthe bridging channel, between the trigger valve and the applied sample.FIG. 7A shows at 110 a schematic view of such a device, and FIG. 7Bshows a system that includes one valve 114, but also a delay unit 112 inadvance of the valve. Once the fluid from spot 116 reaches the valve 114(as shown at t=139 s), the valve 114 is opened, permitting a fluid at118 to migrate with the fluid from the spot 116 to the spot 120. In thisway, the fluids may be mixed at desired times. In a specific biologicalassay, it is preferable to delay the mixing with the next fluid for 1second, 10 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 20minutes, 30 minutes, 1 hour or any desired time period.

In the one such embodiment of sequential-loading steps, a trigger valveand a delay valve are assembled in the array where a selected area,referred to as the reaction spot, can be used as a central point to passfluids sequentially. FIGS. 8A-8B, for example, show a system(schematically at 122 in FIG. 8A) that includes two reserves of fluid(fluid A as shown at 124, and fluid B as shown at 126). After a desiredperiod of time, the fluid B is permitted to mix with fluid A and thenboth reach the absorption terminal 128 together.

The valve may be assembled in two-dimensional (2D) and three-dimensional(3D) assay devices to control biological reactions, such as antibody orreceptor binding, washing and detection steps. A three-dimensionaldevice contains two of more layers of a porous substrate that isuniquely patterned with hydrophobic and hydrophilic regions tofacilitate the preferred directional wicking of the fluid. In addition,a separating layer is placed in between porous layers is impermeable tofluids except in desired regions to transferring fluids from one layerto the other layer.

The transfer region is either a hydrophilic region or a hydrophobicregion. In addition, the shape and the size of the transfer region areselected based upon the desired attributes of the diagnostic device. Ina working three-dimensional device, in a hydrophobic region, the fluidcan pass between the layers from one aligned hydrophilic to anotherhydrophilic region to complete the desired assay. In another embodiment,a fluid is released by a trigger valve contained in a layer above orbelow rather than on the same layer. More precisely, the hydrophobicregion is aligned within the impermeable separating layer in the desiredtransfer region.

FIGS. 9A-9F show illustrative diagrammatic views of three-dimensionalpaper-based microfluidic devices in accordance with various embodimentsof the invention. FIGS. 9A, 9C and 9E show at 130, 132 and 134 variouslayers separated for illustration, and FIGS. 9B, 9D and 9F show topviews of the top layers 140, 142 and 144. FIG. 9G shows at 136 a scannedcross section taken along line 9G-9G of FIG. 9F.

The surfactant is placed either in the above layer or the below layerdepending upon directional flow desired. As shown at 150 in FIG. 10B(FIG. 10A shows a schematic of the valve), a valve is provided usingfirst layer 152 and a second layer 154 separated by a tape that includesa disc and an aperture. The fluid first flows in a direction as shown inthe top view of FIG. 10C, and later flows in a direction as shown at inthe bottom view of FIG. 10C. The fluid flows to the surfactant containedon the bottom layer beneath the hydrophobic disc. The solubilizedsurfactant fluid (158) is able to pass through the hydrophobic disc,thereby opening the valve for fluid to pass to the upper hydrophiliclayer. The thickness of the hydrophobic layer may vary to any thicknessbetween 1 micron to 1000 microns depending up the desired application.Therefore, a detection of the analyte can be performed on a layerseparate from the one where the initial sample was loaded.

As shown at 160 in FIG. 11B (FIG. 11A shows a schematic of the valve),another valve is provided using first layer 162 and a second layer 164separated by a tape that includes multiple discs and apertures.Triggering fluid flows in a direction that opens the valve so that boththe triggering fluid and the gated fluid may be combined.

As shown at 170 in FIG. 12B (FIG. 12A shows a schematic of the valve),another valve is provided using first layer 172 and a second layer 174separated by a tape that includes multiple discs and apertures,including a hydrophilic disc and a hydrophobic disc. The system providesa fluidic delay valve with three channel lengths of delay.

Even more preferably for complex diagnostic assays, the 3D devices maybe designed to operate with two or more fluids as shown in FIGS.13A-13D. As shown at 180 in FIG. 13B (FIG. 13A shows a schematic of thevalve), another valve is provided using first layer 182 and a secondlayer 184 separated by a tape that includes multiple discs andapertures, including a hydrophilic disc and a hydrophobic disc, as wellas an absorbent back layer 186, separated by a further tape A furtherimprovement over the 2D device is the reduction of the diagnostic deviceto approximately the size of a postage stamp, as shown in FIG. 13D. Thecolor graphs at 190, 192, 194 show the color of fluids (yellow and blue)at the spot 196 at times t=3 min, t=8 min and t=12 min.

A sequential-loading system can be used to detect a wide variety ofbiologically desired targets that are represented by the entire orpartial molecule such as a metabolite, peptide, carbohydrate, lipid,nucleic acid or other selective detector molecule that can beselectively bound or interact with a companion detector molecule.Nucleic acid can be either deoxyribonucleic acid (DNA) or ribonucleicacid (RNA). The target analyte will be “trapped” by an antibody,receptor, nucleic acid, chelator, or another molecule capable ofselectively binding or interacting with the target analyte at adetection spot or region. Once the target analyte is bound at thedetection spot, a secondary detector molecule is linked directly orindirectly to a detection agent and is an antibody, receptor, nucleicacid, chelator, or another molecule capable of selectively binding orinteracting with the target analyte. The secondary detector moleculecontains a detection agent such as enzyme/enzyme substrate or gold,fluorescence, phosphorescent, and luminescent tag or marker. Morepreferably, the detection agent is an agent producing a visible colorthat does not require a device to detect the reaction.

The click-chemistry described here or other chemistries can be used tocovalently immobilize to the media a trap molecule that can selectivelybind or interact with the analyte. Examples of covalent bonds includeesters, amides, imines, ethers, carbon-carbon, carbon-nitrogen,carbon-oxygen or oxygen-nitrogen bonds. Alternatively, the trap moleculecan be non-covalently adsorbed to the media provided that thedissociation rate is very low under the conditions used. The trapmolecule can be a metabolite, peptide, carbohydrate, lipid, nucleicacid, molecularly imprinted polymers, inorganic compounds, or anotherselective analyte. Typically, the trap molecule is located in a positionwhere fluids are mixed, and more preferably, sequentially mixed thatprovide the time for incubation and subsequent binding.

The present invention anticipates that in some applications such as withbiological samples, further surface modification of hydrophilic channelsmay be required to reduce nonspecific adsorption of proteins inbiological samples. Specifically, it is desirable to reduce non-specificbinding and interactions between the media substrate and small and largemolecules contained in the clinical or environmental sample. One suchapproach is to replace the hydroxyl-terminated thiol bypolyethylene-glycol (PEG)-terminated thiol as the reactant in the clickchemistry. PEG is a known family of hydrophilic groups that reducenonspecific protein adsorption at water-solid and water-oil interfaces.

In the preferable embodiment of the invention is the application of asample that does not require any prior treatment of the clinical fluidor environmental sample to remove contaminating or obstructing materialsuch as dust, dirt, macroparticles, or microparticles, including cellsor biological aggregates or other biological impurities or removespecific proteins, nucleic acids, inorganic or organic compounds. It isanticipated that a sample may be treated using a variety of protocols toremove the contaminating or obstructing materials, such as subjectingthe fluid to filtering, centrifugation, absorption or other methods. Thepreferred use is for the stand-alone device to incorporate as part ofthe device, a filtering or absorption element to remove or retain theundesired contaminating or obstructing material.

The Examples below are illustrations of representative devices having aselected function but are not limited in scope to their design and thecomponents used in the device such as the combinations of materials usedin the design of the devices, methods for imprinting hydrophobic andhydrophilic regions, and the size, types, materials and position of thevalve or valves using in the devices.

EXAMPLE 1 Patterning of Virtual Barrier Regions on a Paper Matrix

Paper is primarily composed of cellulose fibers that are rich interminal hydroxyl groups. The wettability of a paper sheet was patternedin a two-step reaction (as discussed above with reference to FIG. 1). Inthe first step, cellulose fibers were primed by a trichlorosilane with avinyl terminus (a click chemistry thiol-ene). The condensation reactionbetween the trichlorosilane and the hydroxyl groups grafted the vinylterminus onto the cellulose fibers. Vinyl-terminated trichlorosilanesare known to form hydrophobic monolayers on a variety of surfaces. Asused herein, the vinyl terminus rendered the entire surface and the bulkof the paper sheet hydrophobic.

In the second step, the vinyl terminus further reacted with a thiol inorder to introduce a hydrophilic group (in this scheme, a hydroxylgroup) to the terminus. For this reaction, thiol-ene “click chemistry”was initiated by a photoinitiator (PI) using UV light. Those areas thatwere designated to remain hydrophobic regions were masked to preventphotoinitiation. Therefore, the hydrophilic group was only graftedcovalently in the UV-exposed regions, whereas the masked region remainedhydrophobic. Similar schemes using the thiol-ene chemistry have beenreported for patterning wettability or proteins on surfaces.

Using this scheme, millimeter-scale fluidic channel was fabricated using6-mercapto-1-hexanol as the hydrophilic terminal molecule (see FIG. 2A).In the fabricated device, the start of the channel was round to providefor sample application. In this illustration, dye-containing water(orange in color) was applied to the start region and it was observedthat water was absorbed quickly and spread quickly along the channel.The contrast of color clearly showed the edge of the hydrophilic regionpatterned in the layered hydrophobic paper.

The photomask was printed on a transparency film by an office laserprinter. The profiles of the patterned paper were printed on cellulosepaper. Natural cellulose paper sheet (0.6 mm in thickness, obtained fromInvitrogen, Carlsbad, Calif.) was soaked in solution ofallyltrichlorosilane for 6 hours, rinsed by isopropyl alcohol in anultrasonic bath for 15 min, and air-dried at room temperature in a fumehood. Thiol solution (either 6-mercapto-1-hexanol or MUTEG) was thenpipetted onto the paper sheet. The paper sheet was attached to thephotomask, sandwiched firmly between two cover glasses (1 mm inthickness), and exposed using a UV lamp for 240 seconds (ELC-500,Electro-Lite Corporation, Danbury, Conn.).

During the exposure, the backside of the paper sheet was protected fromUV light. After exposure, the exposed paper sheet was placed on a stackof paper towel, washed by 95% (v:v) of ethanol with 5% (v:v) of water,and dried on a hotplate at 70° C. for an hour. Finally, 0.5 μl of asurfactant solution (Tween-20, 3% in ethanol, w:w), the transfer agentwas deposited to the circle terminal of the hydrophilic transfer region.The layered paper was heated a second time to 70° C. on a hotplate toevaporate the solvent.

To visualize hydrophobicity of the masked, unexposed region, thevertical profile of a 5 μl water droplet was captured resting on thesurface (see FIG. 2B). As a strong evidence of hydrophobicity, thecontact angle of the droplet and the paper surface was found to be118.3±2°. In comparison, the advancing contact angle of a water dropleton a smooth surface packed with self-assembled vinyl termini are in therange of 101° to 107°.

The hydrophilic channels were imaged using a stereoscopic zoommicroscope (Nikon SMZ800) attached with a CCD camera (model SPOT Insight2MP rewire Color Mosaic, Diagnostic Instruments, Sterling Heights,Mich.). The fluidic valves and valve systems were imaged using a digitalsingle-lens reflex camera (Canon).

A water droplet resting on the hydrophobized paper was imaged by astereoscopic zoom microscope with a 45 mirror attached in front of theobjective lens. The water contact angle θ was determined by twogeometric parameters (measured in pixel units), θ=90°±180°/π arcsin h/r,where r and h were the radius of the spherical profile of the dropletand its center distance from the paper surface, respectively. Theuncertainty of contact angle measurement δθ was associated with theuncertainty of each individual geometric parameter, δr and δh, measuredfrom the image. Specifically, δθ was calculated by the root-sum-squareexpression, δθ=[(δθ/δr(δr))²+(δθ/δh(δh))²]^(1/2). In this study, δr andδh were approximately 4 pixel units. Microphotographs were recorded,using the CCD camera and an illuminator (model NI-150, Nikon).

The UV exposure not only produced the hydrophilic region (see FIG. 2A)on the paper surface but it also caused the reaction within the bulk ofthe paper. It was found that the organic solvent that was absorbed inthe paper during fabrication altered the paper from being opaque tobeing semi-transparent. These changes promoted the penetration of UVlight in the bulk. Again, FIG. 2C shows the cross section of thehydrophilic channel along the dotted line in FIG. 2A. The liquid(colored) is distributed from the top to the bottom of the channel.Therefore, the back side of the channel was also wetted.

The uncollimated UV light determined the resolution of hydrophilicpatterns re-produced from the photomask. In FIGS. 3a and 3b , a seriesof hydrophilic channels (wetted by dye-containing water) and hydrophobicgaps are shown with decreasing width, respectively. By using Image J™software, the edges of the channels in the inset in FIG. 3a werehighlighted. A 0.2-mm wide line in a photomask produced a 0.67-mm widechannel shown as the first vertical channel on the right (FIG. 3a ).Line patterns that were less than 0.2 mm in width did not producechannels. Shown in FIG. 3b , a 1.50-mm wide (UV opaque) block in thephotomask produced a functioning hydrophobic gap.

Further reducing the gap width in the photomask resulted in the leakingof water through the gap. FIG. 3c plots the reproduced width of eachchannel measured as the peak-to-peak distance in the profile (inset inFIG. 3C) along the dashed line in FIG. 3A. The reproduced width shiftsfrom the designed width by 0.47 to 0.8 mm. The shift suggests that asafe distance between two lines in a photomask should be longer than 1.6mm, which is consistent with the minimum designed width (1.5 mm) of thefunctioning gap in FIG. 3B.

EXAMPLE 2 Varying the Degree of Hydrophilicity

In addition to the photopatterning, the chemistry described hereinenabled varying the degree of hydrophilicity by the selection a thiolwith other termini. In general, a monolayer constituted byself-assembled, oligo (ethylene glycol)-terminated, pure alkanethiolsexhibits reduced hydrophilicity, compared to a monolayer constituted bysimilarly organized, hydroxyl-terminated, pure alkanethiols. It wasfound that the hydroxyl terminus of 6-mercapto-1-hexanol was disorderlyorganized and projected outwards from cellulose fibers. Theseprojections largely determined the hydrophilicity of the patternedchannels. It was found that by grafting MUTEG (HS(CH₂)₁₁(OCH₂CH₂)₄OH),an alkanethiol with a less hydrophilic tetra-ethylene-glycol (TEG)terminus, the hydrophilicity was significantly reduced within thepatterned region.

Using this MUTEG molecule, an S-shape hydrophilic channel was fabricatedon layered hydrophobic paper (with reference to FIG. 4A). The crosssection of the channel in FIG. 4B was significantly thinner than thatone of FIG. 2C. However, water wicking along the channel could not wetthe reverse side of the layered paper. It was observed that, unlike thehydroxyl-grafted channel, water spread slowly along the TEG-graftedchannel. The slow spread of water in the TEG-grafted paper matrixresulted in the low rate of water absorption when a water droplet wasfed onto the paper surface.

To test water absorption, two 2×2 cm² hydrophilic areas grafted with TEG(see FIG. 4C) or hydroxyl termini (see FIG. 4D) were prepared. The waterabsorption of the TEG-grafted surface was roughly 200-times slower thanthat of the hydroxyl-grafted surface. A 5 μL water droplet was absorbedin 202 seconds into the TEG-grafted compared to one second into thehydroxyl-grafted surface. The reduced rate of water absorption stronglysuggests that TEG-grafted paper is less hydrophilic. It was also foundthat the rate of water adsorption of the hydroxyl-grafted surface wassimilar to that of the native layered paper.

EXAMPLE 3 A Non-Mechanical Microfluidic Valve

A non-mechanical microfluidic valve consists of a group of hydrophilicpatterns wherein a hydrophilic transfer region is separated from asecond hydrophilic region by a hydrophobic channel (see FIG. 5A). Inthis example, a hydrophilic transfer region is a terminal consisting ofa circle and the second hydrophilic region is an open ring surroundingthe hydrophilic transfer region. A hydrophobic channel separates the twohydrophilic regions. A surfactant is deposited within the hydrophilictransfer region. This valve arrangement of hydrophilic patterns and thesurfactant promote wicking only from the hydrophilic transfer region toa second or acceptor hydrophilic region.

FIG. 5B illustrates microscopic events when a water-based fluidapproaches the hydrophobic gap from the hydrophilic transfer region. Thefluid approaching the hydrophilic transfer region dissolves thedeposited surfactant and reaches the joint edges of the hydrophilic andthe hydrophobic regions. The dissolved surfactant molecules adsorb tothe water-air and the water-solid interfaces reducing the associatedsurface tensions. The surfactant molecules also adsorb to thehydrophobic solid-air interface at the locations closest to the contactline that is the edge of the fluid meniscus on the solid surface,resulting in a local increase of solid-air surface tension.

These changes of surface tensions increase the associated spreadingcoefficient, S, and promote fluid spreading along the hydrophobicsurface, thereby “opening” the valve. In contrast, fluid approachingfrom the acceptor hydrophilic region is stopped because this region doesnot contain any surfactant.

The transfer by the surfactant-containing fluid from hydrophilictransfer region to the hydrophilic acceptor region is the criticalfactor in the design of the non-mechanical microfluidic valve. The fluidat the hydrophilic transfer region spreads away from the circle in alldirections. In this example, the particular shape of the hydrophilicacceptor region provides a larger acceptor area to collect and guide thespreading fluid. In other applications, the size and shape of thehydrophilic transfer region and the hydrophilic acceptor region can bevaried for the intended applications and the design used in this exampleis not limited. The dimensions of the hydrophilic transfer region andthe hydrophilic acceptor region are determined principally by theresolution of the photopatterning process and the equipment used.

To validate the functionality of the non-mechanical microfluidic valve,two reverse oriented pairs of a hydrophilic transfer region and ahydrophilic acceptor region were tested in parallel (FIG. 5c ). Ahexagonal input terminal was connected to this pair of valves. Fiftymicroliters of water doped with a food dye was pipetted to the hexagonalterminal (time=0 s) and wicked along the bifurcated path and reachedboth valve pairs (t=21 s). The forwardly configured non-mechanicalmicrofluidic valve (left) promoted wicking through the hydrophobic gap,whereas the reversely configured non-mechanical microfluidic valve(right) stopped the wicking (t=45 s). The final pattern of water (t=106s) confirmed the function of the correctly configured non-mechanicalmicrofluidic valve. In addition, it was observed that the water in thedownstream channel of the left valve penetrated into the hydrophobicregion by a small distance (see the inset of FIG. 5C).

This penetration was caused by the surfactant remaining in the advancingfront of water. It was noted that surfactant depletion occurs whencrossing hydrophobic gaps. The amount of deposited surfactant must beabundant to ensure the complete bridging of water through the gap.However, too much surfactant induces undesirable water spreading indownstream channels. The optimum concentration of deposited surfactantused in this example was found to be 3% Tween-20 in ethanol, weight perweight.

In addition to water, the non-mechanical microfluidic valve was testedusing a biological sample to demonstrate the breadth of applications forclinical diagnostics. In this test, human blood serum, a viscous fluidrich in proteins, was used as the working fluid. FIG. 5D shows bridgingand stopping of blood serum in the forwardly (left) and the reversely(right) configured pairs, respectively. Through testing, it was foundthat the optimal concentration of deposited surfactant had to beincreased to 50% Tween-20 in ethanol, weight per weight.

EXAMPLE 4 A Non-Mechanical Microfluidic Trigger Valve

Non-mechanical microfluidic valves were used as building blocks tocreate more complicated elements such as a trigger valve and a delayvalve.

As an illustration, a trigger valve was required to perform more complexdiagnostic assays, where it is required to mix a sample with a reactivefluid that is released in a timed period. As illustrated in FIG. 6B, afluid sample, serving as a triggering fluid, was used to open the valveat a given time so that a gated fluid containing a reactive agent, suchas an enzyme substrate, or a binding protein, such as an antibody, couldreact before reaching a terminal point.

In this design, a non-mechanical microfluidic valve was placeddownstream of the injection channel to form a trigger valve (shownschematically in FIG. 6A). As shown in FIG. 6B, a triggering fluid(green) was added to the injection region (hexagon, left) that divergedinto two streams after t=89 s. In one direction, the fluid moved towardand reached the microfluidic valve at t=119 s. At t=148 s the triggerfluid bridged the hydrophobic gap thereby allowing the gated fluid(blue) to pass across the hydrophobic gap and mix with the triggerfluid. The mixed trigger and gated fluids then moved together andreached the unwetted absorption terminal at t=292 s.

In this example, 50 μl of the triggering fluid and 100 μl of the gatedfluid were used. A surprising feature was observed in the micrograph att=148 s, namely that the trigger fluid has a preference for wickingtoward the microfluidic valve rather than the absorption terminal. Thisimplies that the amount of the triggering fluid that is deposited in theinjection region is adequate to reach and bridge the microfluidic valve.Thus, it will be primarily the gated fluid that reaches the absorptionterminal. Therefore, the amounts of the triggering and gated fluids canbe adjusted accordingly to ensure that the desired concentration offluids reach the absorption terminal.

EXAMPLE 5 A Non-Mechanical Microfluidic Delay Valve

A hydrophilic transfer region and a hydrophilic acceptor region can bejoined using a bridging channel to form a delay valve (shownschematically in FIG. 7A). The length of the bridging channel determinesthe time delay. Two valves with different delay times were demonstratedwith bridging channels having different lengths resulting in one delaytime being about twice as long as the other (see FIG. 7B: left, 29 mm;right, 63 mm). A fluid volume of 100 μl was applied simultaneously toboth input terminals (t=15 s). The valve with the short bridging channel(left) opened approximately in 3 minutes (t=152 s), whereas the one withthe long bridging channel (right) opened approximately in 9 minutes(t=553 s). After the opening, both advancing fronts of the fluids in thetwo valves moved towards the corresponding absorption terminals. It wasfound that the relation between the delay time, t, and the length of thebridging channel, L, is nonlinear according to Washburn's equation,t/L². This observation confirmed that doubling the length of thebridging channel extended the delay of the valve opening toapproximately 3.7 times.

EXAMPLE 6 More Complex Systems using Multiple Microfluidic Valves

The non-mechanical microfluidic valves are the basic elements that canbe assembled into a more complex diagnostic device, which is able torelease and combine fluids containing different soluble materials. Inthis example, two non-mechanical microfluidic valves, one of which is adelay valve, were used to construct a sequential-loading system. Asshown in the FIG. 8A, the first valve (diode 1) was inserted between theinput terminal of fluid B and the loop of the delay valve. The delayvalve consists of valve 2 (diode 2) and the bridging channel. Valve 1was reversely configured to gate fluid B. A second input terminal forfluid A is added close to the hydrophilic acceptor region of valve 2.All the fluids move towards a large absorption terminal. In this andother examples, all fluidic channels used in the delay devices are shownto have the same width. To reduce the footprint of the devices, achannel's width can be reduced at a certain location to restrict fluidflow, similar to a resistor in electrical circuits. Conversely, thewidth can be expanded to absorb the fluid momentarily before it proceedsalong the channel, providing a function similar to a capacitor in anelectrical circuit.

The system manipulated the fluids to sequentially pass through thereaction spot, shown in FIG. 8B. To initiate operation, Fluid B (blue)and Fluid A (green) were loaded to the corresponding input terminals atthe same time. It was observed that Fluid A diverged into two streams.One stream moved to the acceptor region of valve 2 (t=46 s). The otherstream passed through the reaction spot to the transfer region of valve1. Fluid A opened valve 1, thereby triggering the wicking of Fluid Bthrough it. Because valve 2 was gated, Fluid B moved along the bridgingchannel (t=340 s) to the transfer region of valve 2. Upon opening valve2, Fluid B was able to pass through the reaction spot, replacing Fluid Aand continuing on to the absorption terminal (t=618 s). The length ofthe bridging channel controlled the timing of the sequential loading. Inthis example, the length of the shortcut is roughly one third of that ofthe bridging channel. Therefore, the flow flux of Fluid B through theshortcut path is significantly larger than that in the bridging channel.

The sequential-loading system is particularly useful for biologicalassays. In one such example, one can adapt the multiple valve system fora multistep immunoassay in which target antigens are trapped at thereaction spot in the device and subsequently detected using a secondaryantibody conjugated with a detection indicator.

EXAMPLE 7 3D Paper-Based Fluidic Valves

To improve the flexibility of the sequential-loading system, it wasdemonstrated that layering of patterned paper formed paper-based fluidicdevices with hydrophilic channels in 3D (FIGS. 9A, 9C and 9E). Thepatterned layers shown in FIG. 9A and FIG. 9C were aligned, and stapledtogether at the circular contact spots, forming the two devices shown inFIG. 9B and 9D, respectively. The spots in the top layers were wettedautonomously via channels in the bottom layers, allowing fluids to movevertically and laterally. In FIG. 9B, the device was shown to distributedye-containing water (orange or green in color) from two large spotsinto two 2×2 arrays of small spots. If one changed the pattern of thehydrophilic channels in the bottom layers, the fluid distribution wasaltered according the new configuration. The device shown in FIG. 9ddistributed water into two 4×1 arrays.

It was further demonstrated that if one assembled three layers ofpatterned paper (see FIG. 9E) by the same manner two water streams wereable to cross each other without mixing. As illustrated, the streams(orange or green in color, 100 μl) were fed to the two circular ends,moved along the hydrophilic 3D paths, and crossed over each other forfour times (see FIG. 9F). FIG. 9G shows the cross section along thedotted line in FIG. 9F showing the multiple layer stacking and alignmentof vertical channels.

In another embodiment of the 3D fluidic valve invention, wax printingwas used as an alternative method to define hydrophilic channels onpaper. To assemble a 3D device, wax printed areas were assembled bycutting to the size, aligning the printed areas into a stack, andadhering the combined stack using any number of methods such as tape andglue to prevent the escape of fluid in undesired areas and to preventevaporation of fluids. Once assembled the stack would form the 3Dfluidic valve that regulated fluid flow across the layers.

This design of this 3D fluidic valve is shown in FIG. 10B. The fluidicvalve was fabricated on paper containing the following elements: twolayers containing hydrophilic channels and terminals, a hydrophobiclayer containing hydrophobic permeable gap that separates the channels,and an amount of surfactant deposited onto one of the channel terminalsto facilitate transport across the hydrophobic permeable gap in thedirection desired. The directional flow of the valve is always from theterminal containing surfactant to the terminal that does not contain thesurfactant, similar to a 2D value. In some embodiments of the invention,the complexity of the diagnostic device may require additional layerscontaining hydrophilic channels, hydrophobic layer permeable gaps, andcompanion surfactant in the transfer terminal. The minimal requirementhowever, is three layers.

In the present example, three layers of materials were used to constructthe valve (FIG. 10B): the top and bottom hydrophilic paper layerscontain 1 mm wide channels and terminals defined by wax contours printedon and melted into the fabric of the paper. The middle layer contains ahydrophobic permeable paper disk fitted to a 4 mm diameter hole on adouble-sided and impermeable tape. The paper disk is cut fromtrichlorsilane-treated paper of approximately 140 μm in thickness. Othermaterials with similar permeable hydrophobic properties can substitutethe disk. Paper of other thickness could be used provided that the diskis slightly thicker than the tape to maintain a good contact with theadjacent layers after assembling.

Alternatively, the surfactant can be deposited directly to thehydrophobic disk by applying it as a thin layer of agent that does notpenetrate to the opposite side of the disk. The surfactant is depositedinto and dried onto the terminal of the channel in the bottom layerprior to assembling. The round terminals of the channels are aligned tothe disk forming a permanent assembly with a thickness of approximately0.5 mm. The shape of the aligning terminals of the channels and thehydrophobic disks do not necessarily have to be round. For example,square and rectangular terminals and disks can also be used.

Contours of channels are printed on 200 μm thick filter paper using aXerox Colorqube Printer. The printed paper is placed in a 150° C. ovenfor 40 seconds to allow the wax to melt downwards, which also inks theother side of the paper. The melting broadens the wax lines byapproximately 0.5 mm. The double-sided tape (ACE plastic carpet tape) ispunched with through holes using a 4 μm diameter biopsy punch. Thehydrophobic paper disks are prepared in two steps: 140 μM thick filterpaper is rendered hydrophobic by soaking it in perfluorocarbon oilcontaining 3% (weight percentage) of Allyltricholrosilane for 1 hr,washing it in ethanol, and then drying it on a hotplate at 50° C. Thedisks are cut from the filter using a biopsy punch. The hydrophilicpaper disks are fabricated using the punch and the unmodified filterpaper. Prior to assembling, 0.4 μl of a surfactant solution (Tween-20,2.5% in ethanol, by weight) is deposited to each corresponding onto eachtransfer location followed by drying at room temperature. The devicesare assembled layer by layer.

In the absence of surfactant, the hydrophobic disk prevents a fluid frommoving from the top to the bottom layer. To permit fluid transferbetween hydrophilic layers, a fluid deposited in the loading terminaltravels to the circular transfer area where it dissolves thepre-deposited surfactant acting to reduce the fluid surface tension andfacilitating the transfer of the fluid from the bottom layer to the toplayer through the hydrophobic permeable disk. The diameter of the discarea can be altered to increase or decrease the amount of fluid or thetime to transfer fluid across the permeable hydrophobic disc. While inthis example, a circular area was used, any desired shapes can besubstituted depending upon the required need.

To demonstrate the capability of the device, water containing a dye wasdeposited to one of the two neighboring loading terminals (FIG. 10c ). Adrop of water was deposited to the hexagonal loading terminal on the toplayer without the surfactant in the circular transfer region. The fluiddeposited on the top layer was retained on the top layer throughout theexperiment. In contrast, depositing a drop of water to the bottomloading hexagon terminal transferred to the circular transfer regioncontaining the surfactant and when the surfactant was solubilized thefluid was able to transfer through the hydrophobic permeable region tothe top layer quickly demonstrating the function of the 3D valve.

EXAMPLE 8 A 3D Delayed Trigger Valve

Attaching a channel of varying lengths to the 3D valve formed a triggervalve with a delay. Similar to the 2D valve, the length of the channelwas able to increase or decrease the time of the delay (see FIG. 11C). Atrigger valve is three-terminal component that can stop a fluid untilthe feeding of a secondary triggering fluid. The trigger valve consistsof a valve with a channel that branches off at the valve (see FIG. 11A).The valve and the channel are arranged in such way that the triggeringfluid can move along the channel to short the valve, allowing the gatedfluid to pass. In this example, the trigger valve consisted of threestacked layers (see FIG. 11B). The top layer contains two channels: theshorter channel (gate channel) accepts a gated fluid; the longer andturned channel (trigger channel) accepts a triggering fluid. The roundtransfer regions of the channels are aligned to two paper disks fittedin two separate through holes on the tape, which is the middle layer.The disk aligned with the shorter channel is hydrophobic, whereas theother disk is natively hydrophilic. The bottom layer contains a channelthat visually joins the two channels on the top layer at the verticaldirection. This layer contains a surfactant spot in the round transferregion aligned to the hydrophobic disk in the middle layer.

The delayed trigger valve was demonstrated by adding a drop ofdye-containing water (blue) to the gate channel (see FIG. 11C). Thevalve stopped the fluid transfer to the bottom layer. In contrast, adrop of triggering fluid (yellow) added to the terminal on the top layerspreaded to the bottom layer terminal, containing surfactant, and movedbi-directionally towards the end terminal. Once the surfactant wassolubilized, the fluid was able to penetrate the hydrophobic disk andopened the valve to release the gated fluid (see FIG. 11D). With thevalve open, the gated fluid was released and mixed with the triggeringfluid on the bottom layer and made the channel appear green (FIG. 211,t=16 min).

EXAMPLE 9 A 3D Delayed Trigger Valve for Single Fluids

In certain diagnostics testing, it may be of interest not to release theentire fluid at once but have it delivered after a certain amount oftime. In this instance, the delayed trigger valve can provide this bymerging the terminal of the trigger channel with the gate channel forms(see FIGS. 12A and 12B). The delay creates a time lag when the bulk ofthe fluid is transferred to the other layer. The length, L, of thechannel that is connected to the circular transfer region adjacent tothe hydrophilic non-valve disk, adjusts the timing of the delay.

In this example, it was demonstrated that varying the length of thetiming channel (L=7, 19, 25 mm) created delays with adjusted delay time(FIG. 12C). Each of the three loading regions was loaded withdye-containing water. The shorter length of the timing channel resultsin rapid transfer of the fluid. For example, the fluid tokeapproximately 1.5 minutes to reach the circular transfer region on thetop layer with L=7 mm. In this case, the fluid passed to the outletterminal in 5-9 minutes (FIG. 12C). Other delays were longer dependingupon the length of the channel. For the L=25 mm channel the delay was 12to 17 minutes, while for the L=19 mm the delay was 10 to 13 minutes.

EXAMPLE 10 Sequential Fluidic Valves

For more sophisticated diagnostic testing, it would be essential to passtwo or more fluids through a designated region to provide washing,binding agents, or colorimetric detection.

In this design, two valves were constructed with each having two fluidloading terminals and two companion circular transfer regions where thefluids were passed through a single target spot sequentially (See FIG.13A). Both input terminals were connected to reverse facing. Thefootprint of this circuit was about 24×24 mm² in size, which is similarto that of a postage stamp (see FIG. 13D). The device was operated usingonly two drops of water.

For the sequential-loading circuit, all layers used the filter paperdescribed previously except for Layer 3 that is made of a piece of 300um thick polyester-cellulose cloth (ITW Texwipe, NC, USA).

The construction of 3D diagnostic valve device is shown in FIG. 13B asdiscussed above. The device was constructed with five layers, of whichthree layers are paper and two layers are tape aligned and stackedtogether. Layers 1, 2 and 3 contain two valves whose configuration isshown in the figure. The target spot for the fluid mixing is located atthe center of the top layer. Layer 5 is a 0.8 mm thick paper that isused as a waste absorbent for Layer 3. Layer 4 acts to restrict theabsorption through a single though hole containing a hydrophilic disk.

To demonstrate its diagnostic operation, two fluids containing red orblue dyes were placed to the corresponding loading inlets on Layer 1.Only Fluid A moved to the target spot while some of the fluid was passedthrough the hydrophilic disk to the circular transfer region below. TheFluid A subsequently was split into two directions on Layer 3. In onedirection, Fluid A moved toward the adjacent circular transfer regionfor Fluid B and solubilized the surfactant to open the valve for FluidB. Once the valve is open, Fluid B is transfer to Layer 3 and mixes withFluid A in the channel on Layer 3. The mixed fluids stream traveledthrough a delay channel on Layer 3 toward the second circular transferregion beneath the hydrophobic valve adjoined to Fluid A in a secondcircular region in the loading terminal.

Once the valve is open, Fluid B has an alternate and shorter route toreach the absorption pad. The newly openly faster route passes thecenter spot on Layer 1 (dashed line), whereas the second slower routeremains within Layer 3 (dotted line).

By feeding two dye-containing fluids (yellow and blue), it wasdemonstrated that the color of the center spot on the top layer changesfrom yellow, to green (upon mixing), and finally blue (FIG. 13C). Theimages of the device were recorded at three different times and theintensity of the center spot was measured for each image. Themeasurement of the intensity at the spot also shows the fading of yellowcolor and the brightening of blue color over time. These resultsconfirmed the sequential-loading function of the device.

EXAMPLE 11 More Complex Sequential Fluidic Valves

It should be noted that there exists numerous combinations to construct3D sequential-loading devices shown in FIG. 13B, depending upon the userequired for the diagnostic assay. For example, FIG. 14 shows atmicrofluidic system that includes three layers 202, 204, 206 separatedby tapes that each include apertures, hydrophilic discs and hydrophobicdiscs, and an absorbing bottom layer 208 as shown. The 3D assemblyincorporates 7 layers of material. This assembly incorporates 2additional layers, including a paper and tape layers, to distributefluidic channels across the added layers. In this design, the length ofthe channels could be reduced in each paper layer, leading to furtherminiaturization of the footprint.

In yet other examples, the fluidic valve technology is capable tomanipulate more than two fluids to pass a designated spot in thecircuit. In FIGS. 15A and 15B two examples of sequential valve diagramsare shown at 210 and 212 in schematic form. In the first diagram, FIG.15B, three fluids are timed to exchange in the target circular spot 216.The triangles represent the directional of the valve. The zig-zagchannels represent delay channels in which flow rate of a fluid isslowed relative to others channels in the device. In this device, thesequential order of fluids being passed through the target spot is FluidA, then Fluid B, then mixture of Fluid B and C. In another example, theorder was altered so that the sequential order of fluids being passedthrough the target spot (214 or 216) is Fluid A, then Fluid B, thenmixture of Fluid A and C.

The simple eloquence of the fluidic valve technology is that it readilycan be adapted to handle a variety of fluidic exchanges depending uponthe desired diagnostic application, such as sample fluids, detectionbinding agents, colorimetric substrates, washing fluids, enzymeactivators or inhibitors, and any other materials contained in a fluid.

EXAMPLE 12 Fluid Reservoirs

Fluidic circuits required sufficient fluid volumes to perform thedesired function and in certain cases, such as washing fluids, mayrequire larger volumes to feed into their inlet terminals. The feedingprocess can be achieved by using reservoirs built into the fluidicdevice that carry the reagents. Folding the paper device on apredetermined axis and matching the reservoirs with fluid inletterminals on the paper circuit can assemble these devices with largerreservoirs. The reservoirs themselves are designed to allow for inletflow regardless of the direction of the force of gravity and do notallow for fluid flow before the reservoirs are connected to the papercircuit.

As illustrated in the system 220 of FIG. 16A, thin walled plasticreservoirs 222, 224 may be used to hold reagent fluids and are insertedinto slots 1 and 2 (226, 228) on side B of the paper chip 230. Toprevent leaking during storage, a thin, impermeable membrane initiallycovers the reservoirs. Before the device can be used, the membrane isremoved leaving pre-wetted reservoir pads that will be lined up withfluid inlet terminals during the folding action. The reservoir pads willprevent reagents from pouring out but allow for capillary flow into thepaper fluid inlets. As shown in FIG. 16B, a sponge 232 is used to guidefluid within each reservoir to the connection with the paper circuit insuch a way that the device can be held in any direction and still havecapillary flow out of the chamber regardless of the direction ofgravity.

The reservoirs that hold the reagents for a diagnostic assay have twomembranes over the opening. The outer layer is an impermeable membranethat is used as protection against evaporation, spillage, andcontamination during storage. This membrane is removed immediatelybefore use of the device. The second membrane prevents the fluid reagentfrom pouring out of the reservoir, yet has a pore size, which allows forcapillary flow when in contact with the fluid device (FIG. 16B). Thismembrane also adds some thickness to ensure full contact between thereservoir and the diagnostic device. To counteract the settling of thefluid reagent because of gravity, an artificial sponge is used on theinside of each reservoir to guide the reagent out of the reservoir.

EXAMPLE 13 Device Architecture for Large Volume Fluid Reservoirs

In yet other embodiments to increase the functionality of the diagnosticdevice, another design is shown for passing a large quantity of samplethrough a target spot. A folding structure has been developed toaccomplish this in a 3D sequential-loading device. This foldingstructure is an extension of the standard two input sequential-loadingcircuit, by integrating a movable detection target spot. For example,FIGS. 17A and 17B show a five layer system 240 that includes layers 242and 244, as well as tape layers and an absorbent layer 246. The tapelayers include apertures and hydrophobiuc discs 250 and hydrophilicdiscs 248.

Prior to folding the microfluidic channels are discontinuous. Whendetection target spot and reservoirs are folded over into the detectionposition, the diagnostic device becomes functional by allowed thetransfer of fluids to pass through the target spot is designed manner.

EXAMPLE 14 Alternative Design Enabling Functionality

In yet alternative example of a simple two-step diagnostic device thatbecomes functional upon folding is shown in FIGS. 18A and 18B in which afirst layer portion 262 is folded onto a second layer portion 266, andthen positioned above another layer 264. The layers include circuitswith valves in accordance with the invention as shown to provide afluidic valve with a coupled reservoir. Initially the top layer isparallel to the remaining layers (see FIG. 18A). For example, a fluidsample containing a antigen to be detected is placed in the inletterminal to the left of the target spot. The fluid sample flows throughthe hydrophilic channel, and up and over the detection target spotcontaining dried immobilized capture antibody, and then down to theabsorption pad. The absorption pad should have excess volume capacity toabsorb all fluids that are containing in the device.

To enable the device, after the right half of the top layer will befolded 180° manually (see FIG. 18B). Successfully un-bridging thedetection ‘spot’ from the sample while the target spot will then bridgethe channel of the circuit, allowing a detection antibody to passthrough the spot, as previously described herein. The folding actionsimultaneously makes contact between the detection antibody andsubstrate, Fluids A and B, respectively, to the two inlets on the leftside of the circuit.

EXAMPLE 15 A Diagnostic Fluid Device Using an Enzyme Detection System

A diagnostic fluid device using fluorescence detection, such as GFP, iswell known in the art. In other embodiments, a visible detection usinggold or an enzyme based detection approaches would provide a visualassessment without a highly specialized detector. This exampledemonstrates the use of an enzyme linked detection system similar tothat used in a standardized ELISA assay but performed in a paper 3Dfluid device. The paper based ELISA is denoted as PELISA.

The PELISA device was fabricated by patterning hydrophobic wax inhydrophilic sheets of paper to create channels as described herein.After patterning, the layers were stacked to form a sequential-loadingdevice. For this demonstration, the antigen to be detected was rabbitIgG, as a model analyte. The concentrations of rabbit IgG to be detectedranged from 1 μg/mL to 1 mg/mL.

Colorimetric assays are well known for usage in situations lackingexpensive plate readers or fluorescence scanners. There are numerousenzyme/substrate pairs used in established ELISA to create a visibleproduct. Alkaline phosphatase was used in this example as the detectionenzyme with its substrate, BCIP/NBT (5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium). This combination was selectedbecause the color variation changes from yellow to purple, therebyproducing an excellent distinction with the white background of thepaper.

As a proof of concept, a PELISA 3D device was loaded in the detectionregion with 0.5 μL of sample containing a concentration of 100 ug/mlrabbit IgG. An antigen, 50 ng of rabbit IgG was immobilized byhydrophilic interaction on the detection region. In addition, thechannel is coated with BSA to prevent nonspecific adsorption ofproteins.

The device's gated terminal was loaded with 200 μL of substrate at thegated terminal. FIG. 19A, for example, shows a system 270 that providesfor fluidic valve using enzymatic detection, and FIG. 19B shows fivesuch detection arrays 280. To activate the device, 100 μL ofenzyme-conjugated detection antibody was added to inlet terminal (shownat 272). The sample fluid passed through the detection region, allowingthe enzyme-conjugated detection antibody to react with the bound rabbitIgG. The fluid continued through a non-valve hydrophilic disk into thedelay channel in the layer underneath. The fluid in the delay channelflowed to a transfer region containing surfactant and opened the valvecontaining the gated substrate. Once opened the substrate traveled backthrough the channel and passed through the detection region. The boundenzyme-conjugated detection antibody subsequently reacted with thesubstrate to produce a visible purple color.

In FIG. 19B, four examples of the 100 ug/ml sample are shown. Samples ofless or more rabbit IgG protein produced a corresponding lesser orgreater purple product. This PELISA system can be used with any of thepreviously described sequential valve devices, though not limited tothose specific devices described herein.

Those skilled in the art will appreciate that numerous modifications andvariations may be made to the above disclosed embodiments withoutdeparting form the spirit and scope of the invention.

What is claimed is:
 1. A microfluidic valve system comprising a matrix,a hydrophilic acceptor region, a hydrophilic transfer region, and ahydrophobic gap between the acceptor region and the transfer region,wherein said system further includes a wetting fluid that when added tothe transfer region, solubilizes a transfer agent at the hydrophilictransfer region and permits the wetting fluid to span the hydrophobicgap to the hydrophilic acceptor region.
 2. The microfluidic valve systemas claimed in claim 1, wherein said transfer agent is separated from thehydrophilic acceptor region by the hydrophobic gap.
 3. The microfluidicvalve system as claimed in claim 1, wherein the transfer and acceptorregions include hydrophilic porous substrates.
 4. The microfluidic valvesystem as claimed in claim 3, wherein said hydrophilic porous substratesare selected from a group consisting of cellulose, glass microfibers,cotton, wool, silk, and combinations thereof.
 5. The microfluidic valvesystem as claimed in claim 1, wherein the hydrophobic gap includes ahydrophobic porous substrate.
 6. The microfluidic valve system asclaimed in claim 5, wherein said hydrophobic porous substrate isselected from a group consisting of polyvinylidene fluoride, nylon,nitrocellulose, polytetrafluoroethylene, mixed cellulose ester, andcombinations thereof, as well as cellulose filter paper renderedhydrophobic by soaking in perfluorocarbon oil containing 3% by weight ofAllytrichlorosilane.
 7. The microfluidic valve system as claimed inclaim 1, wherein said transfer agent is a surfactant.
 8. Themicrofluidic valve system as claimed in claim 7, wherein said surfactantis a non-ionic surfactant selected from the group consisting ofpolyoxyethylene glycol alkyl ether, polyoxypropylene glycol, alkylether, polyoxyethylene glycol sorbitan alkyl ester (polysorbate),polysorbate 20 (Tween 20), polyoxyethylene glycol octylphenol ether(Triton X-100), glycerin, polyoxyethylene glycol alkylphenol ether,polyvinyl alcohol, polysorbate, glycerol alkyl ester,polyvinylpyrrolidone, polyethylene glycol, and glucoside alkyl ether. 9.The microfluidic valve system as claimed in claim 1, wherein said systemincludes a channel of a desired length between an input fluid terminaland the hydrophilic transfer region to provide a delay.
 10. Themicrofluidic valve system as claimed in claim 1, wherein said systemincludes a valve that functions as a trigger valve, providing a releaseof a fluid when opened.
 11. The microfluidic valve system as claimed inclaim 1, wherein the hydrophobic gap permeates a thickness ofhydrophilic media defining a hydrophobic channel separating thehydrophilic transfer region containing the transfer agent and thehydrophilic acceptor region.
 12. The system of claim 1, whereinhydrophilic and hydrophobic regions are patterned on a medium usingmaterials to create a physical solid barrier, a non-physical hydrophobicbarrier or a combination thereof.
 13. The system of claim 12, whereinthe physical solid barrier is deposited onto the matrix and the materialis selected from a group consisting of polystrene, PDMS, wax andphotoresist.
 14. The system of claim 12, wherein the non-physicalhydrophobic barrier is made by covalently modifying the terminal groupsof the matrix into hydrophobic and hydrophilic regions.
 15. The systemof claim 10, wherein the fluid is selected from a group consisting of asample, buffer, antibody, enzyme, enzyme substrate, detection agent anda combination thereof.
 16. The system of claim 15, wherein the detectionagent is selected from a group consisting of a gold, fluorescence,phosphorescent, and luminescent tag or marker.
 17. The system of claim15, wherein the sample contains an analyte to be detected, wherein theanalyte is selected from a group consisting of a peptide, protein,nucleic acid, fatty acid, metabolite, organic compound, inorganiccompound or a combination thereof.
 18. The system of claim 1, whereinthe matrix contains a plurality of valves selected from a groupconsisting of microfluidic valve, delay valve and trigger valve.
 19. Thesystem of claim 18, wherein the plurality of valves is placed in asequence to allow the transfer and mixing of a plurality of fluids. 20.The system of claim 19, wherein the plurality of sequential valves isconfigured on a single matrix or a plurality of matrixes in a threedimensional (3D) array.
 21. The system of claim 19, wherein theplurality of sequential valves is configured on the plurality ofmatrixes, and wherein the matrixes in the 3D array are separated by animpermeable layer that prevents fluid transfer.
 22. The system of claim21, wherein the impermeable layer contains a porous hydrophobic regionand a porous hydrophilic region.
 23. The system of claim 22, wherein theporous hydrophobic region is the hydrophobic gap in the microfluidicvalve.
 24. The system of claim 21, wherein the microfluidic valve, whenopened, transfers the fluid between the plurality of matrixes, whereinone matrix has the hydrophilic transfer region and another matrix hasthe hydrophilic acceptor region.